Frans_M_Everaerts_Isotachophoresis_378342.pdf

EXAMPLES
II
4 3
Fig.5.11. Isotachopherograms for the separation of phosphate and salicylate in various operational
systems, to show the difference in separation according to pK values and according to mobilities.
The centre isotachopherogram is as described in Fig.5.10 (centre). AU other conditions are as in
Fig.5.10, except for the operational systems. Left-hand isotachopherogram: leading electrolyte,
0.01 N hydrochloric acid (pro analysi grade) adjusted to pH 3.2 by the addition of p-alanine.
Right-hand isotachopherogram: leading electrolyte, 0.01 N hydrochloric acid (pro analysi grade)
adjusted to pH 7 by the addition of imidazole. In both instances, a complete separation could be
achieved. t = Time; R = increasing resistance; A = increasing W absorption. 1 = Chloride; 2 = phosphate;
3 = salicylate; 4 = glutamate.
the ionic species to be separated. We could use a leading electrolyte with a pH, of about
5, but there is then a problem. When the pH in the zones is about 5, the ionic species
with pK values of 2 and 3 have nearly identical effective mobilities and they cannot be
separated. For the ionic species with pK values of 7 and 8, the effective mobility is rather
low and it is then preferable to carry out the separation in two runs.
In the first run, we use a leading electrolyte with a pH of, e.g. ,3.5, so that we can
easily separate the anionic species with pK values of 2,3 and 5. Further, a terminating
ionic species is chosen such that its effective mobility is higher than those of the ionic
species with pK values of 7 and 8.
In the second run, we take a leading electrolyte with a pH of about 6.5 and we can
easily separate the anionic species with pK values of 5,7 and 8 (other ionic species
will generally form a mixed zone). With the two runs, we can thud separate the whole
mixture. In such a separation, we can speak of a ‘combination of systems’. As with
differences in pK values, we also can combine electrolyte systems with the aim of using
different properties such as complex formation for the separation of metal ions and
amino acids (see section 13.1.4).
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